Inexpensive, gentle, highly specific, robust, and rapid techniques are required for the separation and isolation of macromolecules and whole cells from complex mixtures. The most prevalent separation techniques include filtration, centrifugation, extraction, adsorption, chromatography, precipitation, electrophoresis, isopycnic sedimentation, and isokinetic gradients. The type of separation technique suitable for a system depends on the nature of biological molecule and complexity of the media from which it is to be isolated. All of these bioseparation techniques rely on the physical (size, density, shape) or chemical (charge, solubility) differences between biological macromolecules to effect the separation. In many biological mixtures, the physical and chemical characteristics of the species to be separated are often very similar. In addition, currently existing procedures for isolations of biological molecules cause considerable shear-induced damage to the target, especially, in bioassays that require several rinse steps, Pretlow, T. G., Pretlow, T. P., Cell Separationxe2x80x94Methods and Selected Applications, Academic Press, New York, 1-5 (1982-1987).
Techniques that rely on specific chemical linkages (affinity purification) rather than physical differences open up the possibilities for isolation of macromolecules that are difficult to separate using other methods. In colloidal magnetic affinity separation schemes, the substrate consists of magnetic particles distributed uniformly throughout the mixture-containing solution, enhancing the probability of substrate-target contact. Highly specific linkages between the ligand-coated superparamagnetic particles and target materials (with surface ligates) are used to preferentially magnetize the targets. Steady magnetic field gradients are then employed to immobilize and isolate these targets. Current use of magnetic particles for cell separations, Hancock J. P., Kemshead, J. T., Journal Immunological. Methods, 164 51 (1993); Jacobs, n., Moutschen, M. P., Boniver, J., Greimers, R., Schaaf-Lafontaine, N., Res. Immunology, 144, 141 (1993); Funderud, S., Erikstein, B., Asheim, H. C., Nustad, K., Stokke, T., Blomhoff, H. K., Holte, H., Smeland, B., Eur. J. Immunnol., 20, 201 (1993); Schmitt, D. A., Hanau, D., Cazenave, J. P., J. Immunogenet., 16, 157 (1989); Aardingham, J. E., Kotasek, D., Farmer, B., Butler, R. N., Mi, J. X., Sage, R. E., Dobrovic, A., Cancer Research, 53, 3455, (1993); Kvalheim, G., Fjeld, J. G., Pil, A., Funderud, S., Ugelstad, J., Fodstad, O., Nustad, K., Bone Marrow Transpolant, 4, 567, (1989); Skjonsberg, C., Kill Blomhoff, H., Gaudernack, G., Funderud, S., Beiske, K., Smeland, E. B., Scand. J. Immunology, 31, 567, (1990); Drancourt, M., George, F., Brouque, P., Sampol, J., Raoult, D., Journal Clinical Microbiology, 30, 2118 (1992); Dairkee, S., Heid, H. W., In Vitro Cell Dev. Biol., 29A, 427, (1993); Tanaka H., Ishida, Y., Kaneko, T., Matsumoto, N., Br. J. Haematol, 73, 18, (1989); Cottler-Fox, M., Bazar, L. S., Deeg, H. J., Prog. Clin. Biol. Res., 333, 277, (1990); Brinchmann, J. E., Gaudernack, G., Thorssy, E., Jonassen, T. O., Vartdal, F., J. Virol. Methods, 25, 293, (1989); Kemshead, J. T., Elsom, G., Patel, K., Prog. Clin. Biol. Res., 333, 235 (1990); and Belter, P. A., Cussler, E. L., Hu, W. S., Bioseparations: Downstream processing for biotechnology, John Wiley and sons, New York, (1988); for other biological macromolecules; Nunez, L., Kaminski, M. D., American Chemical Society: Chemtech, 41, (1988); and for metals; Sonti, S. V., Ph. D. Thesis, University of Rhode Island, (1995) have been reviewed extensively. Surfaces of particles containing magnetic cores can be derivatized with a large repertoire of functional groups, making this idea potentially feasible for many unexplored applications.
However, several important limitations of currently available technology have restricted the applicability of colloidal magnetic separation. These include inadequate specificity (often caused by diffusion-limited attachment of targets to the magnetic particlesxe2x80x94larger targets in a suspension arrive at the affinity surface slower than the smaller, non-target macromolecules) and the long time necessary to achieve the required degree of separation if target viability has to be maintained (agitation results in damaging shear forces). Because magnetic particles have specific gravities that are significantly larger than water or aqueous salt solutions, they have a tendency to sediment, and must be kept suspended by Brownian motion. This severely restricts their size, and, because the magnetic susceptibility scales with particle volume, requires use of high magnetic field gradients to mobilize them through the surrounding liquid phase. Furthermore, most existing devices operate in the batch mode. This limits throughput and leads to large amounts of down time. The economics of this procedure have made it useful only for very high value products and processes such as cell sorting, DNA purification, protein capture, and microorganism isolation; Haukanes, B. I., Kvam, Bio/Technology, 11, 60, 1993); and Olsvik, O., Popovic, T., Skjerve, E., Cudjoe, K., Hornes, E., Ugelstad, J., Uhlen, M., Clin. Micr. Rev., 7, 43, (1994). Technological advances that speed up this process while simultaneously enhancing target specificity can make a significant impact to this burgeoning area.
We have discovered a new flow-through, multiunit device that potentially removes many of these limitations, resulting in high specificity and reduced separation time.
Broadly the invention is a device (system) and method for the magnetic separation of target particles (macromolecules) from a mixture. Biotin is bound to a target particle. Magnetics beads labeled with avidin or streptlavidin are mixed with the target particles. The avidin or streptlavidin binds to the biotin and the bound complex is magnetically separated from the mixture.
The invention embodies a flow-through multi magnetic-unit device comprising a slowly rotating horizontal chamber designed for a colloidal magnetic affinity separation process. Each magnetic unit consists of an alternating current carrying solenoid surrounding the chamber, and a pair of permanent magnets located downstream from the solenoid, that rotate with the chamber. The chamber rotation simulates a low gravity environment, severely attenuating any sedimentation of non-neutrally buoyant magnetic particles as well as feed, thus promoting good particle-target contact throughout the chamber volume. The oscillating magnetic field gradient produced by the solenoid introduces translational and rotary microparticle oscillations, enhancing mixing, while the permanent magnets immobilize the targets on the chamber walls.
The preferred embodiment is described with a feed system comprising xcx9c50% mixture of biotinylated latex beads (target) and non-functionalized latex beads (non-target) to support that the target particles can be captured and separated from the non-target particles. A maximum separation capture efficiency of 60% and a separation factor of xcx9c18.28 with purity as high as 95% has been achieved.